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protocol_223.ann
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T1 Action 60 63 Add
E1 Action:T1 Acts-on:T3 Site:T5
T2 Amount 64 68 1 mL
T3 Reagent 69 83 TRIzol Reagent
R1 Measure Arg1:T3 Arg2:T2
T4 Amount 87 96 50-100 mg
T5 Reagent 100 124 frozen Drosophila tissue
T6 Size 130 136 1.5 mL
T7 Location 137 157 microcentrifuge tube
T8 Action 163 173 homogenize
E2 Action:T8
T9 Device 191 218 a disposable plastic pestle
R2 Measure Arg1:T5 Arg2:T4
R3 Measure Arg1:T7 Arg2:T6
R4 Meronym Arg1:T5 Arg2:T7
T10 Action 220 229 Centifuge
E3 Action:T10 Acts-on:T11
T11 Reagent 234 240 sample
T12 Speed 244 254 12,000 x g
T13 Time 259 269 10 minutes
T14 Temperature 273 276 4˚C
R5 Setting Arg1:E3 Arg2:T12
R6 Setting Arg1:E3 Arg2:T13
R7 Setting Arg1:E3 Arg2:T14
T15 Reagent 279 285 Pellet
T16 Reagent 295 298 ECM
T17 Reagent 300 315 polysaccharides
T18 Reagent 321 346 high molecular weight DNA
R8 Meronym Arg1:T16 Arg2:T15
R9 Meronym Arg1:T17 Arg2:T15
R10 Meronym Arg1:T18 Arg2:T15
T19 Reagent 348 359 supernatant
T20 Reagent 373 376 RNA
R11 Meronym Arg1:T20 Arg2:T19
T21 Action 446 452 Remove
E4 Action:T21 Acts-on:T23
T22 Action 457 464 discard
E5 Action:T22 Acts-on:T23
T23 Reagent 465 480 the fatty layer
T24 Action 482 490 Transfer
E6 Action:T24 Acts-on:T29 Site:T30
T25 Reagent 543 549 sample
T26 Action 530 538 Incubate
E7 Action:T26 Acts-on:T25
T27 Time 554 563 5 minutes
T28 Temperature 567 583 room temperature
T29 Reagent 495 514 cleared supernatant
T30 Location 520 528 new tube
R12 Setting Arg1:E7 Arg2:T27
R13 Setting Arg1:E7 Arg2:T28
T31 Action 585 588 Add
E8 Action:T31 Acts-on:T33
T32 Amount 589 595 0.2 mL
T33 Reagent 599 609 chloroform
T34 Action 615 618 cap
E9 Action:T34 Acts-on:T35
T35 Location 623 627 tube
T36 Modifier 628 636 securely
R14 Mod-Link Arg1:E9 Arg2:T36
R15 Measure Arg1:T33 Arg2:T32
T37 Action 638 643 Shake
E10 Action:T37 Acts-on:T38
T38 Location 648 652 tube
T39 Modifier 653 664 vigorously
T40 Modifier 664 671 by hand
T41 Time 676 686 15 seconds
R16 Mod-Link Arg1:E10 Arg2:T39
R17 Mod-Link Arg1:E10 Arg2:T40
R18 Setting Arg1:E10 Arg2:T41
T42 Action 688 696 Incubate
E11 Action:T42
T43 Time 701 712 2-3 minutes
T44 Temperature 716 732 room temperature
T45 Action 734 744 Centrifuge
E12 Action:T45 Acts-on:T46
T46 Reagent 749 755 sample
T47 Speed 759 769 12,000 x g
T48 Time 774 784 15 minutes
T49 Temperature 788 791 4˚C
R19 Setting Arg1:E12 Arg2:T47
R20 Setting Arg1:E12 Arg2:T48
R21 Setting Arg1:E12 Arg2:T49
T50 Action 977 983 Remove
E13 Action:T50 Acts-on:T51
T51 Reagent 988 1001 aqueous phase
T52 Reagent 1009 1015 sample
R22 Meronym Arg1:T51 Arg2:T52
T53 Action 1019 1026 angling
E14 Action:T53 Acts-on:T54
T54 Location 1031 1035 tube
T55 Generic-Measure 1036 1042 at 45˚
T56 Action 1047 1056;1070 1073 pipetting out
E15 Action:T56 Acts-on:T57
T57 Reagent 1061 1069 solution
R23 Setting Arg1:E14 Arg2:T55
T58 Action 1075 1080 Place
E16 Action:T58 Acts-on:T59 Site:T60
T59 Reagent 1085 1098 aqueous phase
T60 Location 1106 1114 new tube
T61 Action 1248 1253 saved
E17 Action:T61 Acts-on:T62
T62 Reagent 1189 1240 The interphase and organic phenol-chloroform phases
T63 Time 1301 1310 overnight
T64 Temperature 1314 1317 4˚C
R24 Setting Arg1:E17 Arg2:T63
R25 Setting Arg1:E17 Arg2:T64
T65 Action 1450 1453 Add
E18 Action:T65 Acts-on:T68 Site:T69
T66 Amount 1454 1460 0.5 mL
T67 Concentration 1464 1468 100%
T68 Reagent 1469 1480 isopropanol
T69 Reagent 1488 1501 aqueous phase
R26 Measure Arg1:T68 Arg2:T67
R27 Measure Arg1:T68 Arg2:T66
T70 Action 1503 1511 Incubate
E19 Action:T70 Acts-on:T71
T71 Reagent 1512 1518 sample
T72 Temperature 1522 1538 room temperature
T73 Time 1543 1553 10 minutes
R28 Setting Arg1:E19 Arg2:T72
R29 Setting Arg1:E19 Arg2:T73
T74 Action 1555 1565 Centrifuge
E20 Action:T74
T75 Speed 1569 1579 12,000 x g
T76 Time 1584 1594 10 minutes
T77 Temperature 1598 1601 4˚C
T78 Action 1710 1716 Remove
E21 Action:T78 Acts-on:T79 Site:T80 Creates:T81
T79 Reagent 1721 1732 supernatant
T80 Location 1742 1746 tube
T81 Reagent 1756 1770 the RNA pellet
T82 Action 1772 1776 Wash
E22 Action:T82 Acts-on:T83 Using:T86
T83 Reagent 1781 1787 pellet
T84 Amount 1793 1797 1 mL
T85 Concentration 1798 1801 75%
T86 Reagent 1802 1809 ethanol
R30 Measure Arg1:T86 Arg2:T85
R31 Measure Arg1:T86 Arg2:T84
T87 Action 1823 1829 stored
E23 Action:T87 Acts-on:T88 Site:T90
T88 Reagent 1812 1815 RNA
T89 Concentration 1833 1836 75%
T90 Reagent 1837 1844 ethanol
T91 Time 1854 1860 1 year
T92 Temperature 1864 1869 -20˚C
T93 Time 1883 1889 1 week
T94 Temperature 1893 1896 4˚C
R32 Or Arg1:T91 Arg2:T93
R33 Or Arg1:T92 Arg2:T94
R34 Measure Arg1:T90 Arg2:T89
R35 Setting Arg1:E23 Arg2:T91
R36 Setting Arg1:E23 Arg2:T92
T95 Action 1906 1912 vortex
E24 Action:T95 Acts-on:T96
T96 Reagent 1917 1923 sample
T97 Modifier 1898 1906 Briefly
R37 Mod-Link Arg1:E24 Arg2:T97
T98 Action 1924 1934 Centrifuge
E25 Action:T98 Acts-on:T99
T99 Location 1939 1943 tube
T100 Speed 1947 1956 7,500 x g
T101 Time 1961 1970 5 minutes
T102 Temperature 1974 1977 4˚C
T103 Action 1978 1985 Discard
E26 Action:T103 Acts-on:T104
T104 Reagent 1990 1994 wash
R38 Setting Arg1:E25 Arg2:T100
R39 Setting Arg1:E25 Arg2:T101
R40 Setting Arg1:E25 Arg2:T102
T105 Action 1996 2002 Vacuum
E27 Action:T105 Acts-on:T107
T106 Action 2006 2013 air dry
E28 Action:T106 Acts-on:T107
T107 Reagent 2018 2028 RNA pellet
T108 Time 2033 2045 5-10 minutes
R41 Setting Arg1:E27 Arg2:T108
R42 Setting Arg1:E28 Arg2:T108
T109 Action 2133 2143 Resuspend
E29 Action:T109 Acts-on:T110 Site:T111
T110 Reagent 2147 2157 RNA pellet
T111 Reagent 2161 2177 RNase-free water
T112 Action 2181 2188 passing
E30 Action:T112 Acts-on:T113 Count:T115 Using:T116
T113 Reagent 2193 2201 solution
T114 Modifier 2202 2213 up and down
T115 Numerical 2214 2227 several times
T116 Device 2238 2249 pipette tip
R43 Mod-Link Arg1:E30 Arg2:T114
T117 Action 2251 2259 Incubate
E31 Action:T117 Site:T118
T118 Location 2265 2275 water bath
T119 Location 2279 2293 heat block set
T120 Temperature 2297 2304 55-60˚C
T121 Time 2309 2322 10-15 minutes
R44 Or Arg1:T118 Arg2:T119
R45 Setting Arg1:E31 Arg2:T120
R46 Setting Arg1:E31 Arg2:T121
T122 Action 2324 2358 Proceed to downstream applications
E32 Action:T122
T123 Action 2406 2411 store
E33 Action:T123
T124 Temperature 2415 2420 -70˚C
R47 Setting Arg1:E33 Arg2:T124